US8951957B2 - Delivery agent - Google Patents

Delivery agent Download PDF

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US8951957B2
US8951957B2 US12/901,840 US90184010A US8951957B2 US 8951957 B2 US8951957 B2 US 8951957B2 US 90184010 A US90184010 A US 90184010A US 8951957 B2 US8951957 B2 US 8951957B2
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protein
transfection
peptide
cells
agent
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US20110086794A1 (en
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Arunas Lagunavicius
Lolita Zaliauskiene
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Thermo Fisher Scientific Baltics UAB
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Fermentas UAB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • A61K47/48015—
    • A61K47/48192—
    • A61K47/48223—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/605—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the macromolecule containing phosphorus in the main chain, e.g. poly-phosphazene

Definitions

  • the present invention relates to a composition for delivering a peptide or protein into a cell, a kit for delivering a peptide or protein into a cell and a method therefor, as well as uses of the compositions and kits.
  • Efficient foreign protein delivery into living cells can completely bypass transcription-translation processes related to gene expression, reducing the time required for target protein synthesis from days to hours.
  • the ideal peptide/protein (P/P) delivery method/agent into eukaryotic cells should meet three major criteria: i) it should efficiently bring P/P into the cells; ii) it should protect P/P against denaturation and degradation; iii) the method itself should be non-toxic to the host cells.
  • P/P delivery or transfection techniques include physical delivery methods, such as microinjection, electroporation, and methods based on chemical transfection agents of different origin that facilitate protein delivery into the cell.
  • a general characteristic of a protein delivery agent is its ability to form positively charged complexes with target P/P, which are capable of attaching themselves to negatively charged cell surfaces, crossing through the plasma membrane and delivering the captured protein into the cell.
  • the transfection agent stabilizes the protein, protects it from degradation and preserves its natural characteristics during transfection.
  • Genlantis/ BioPorter P/P forms non-covalent WO03095641A1 Gene Therapy complexes with Systems cationic lipids mixture.
  • Pierce Pro-Ject P/P forms non-covalent complexes with cationic lipids mixture.
  • Stratagene BioTrek P/P forms non-covalent complexes with lipid formulated reagent.
  • Synvolux SAINT PhD P/P forms non-covalent EP0755924B1 Therapeutics complexes with cationic pyridinium amphiphile and a helper lipid.
  • ProFect-P1 forms non-covalent Systems
  • NEB TransPass P P/P forms non-covalent complexes with non-lipid polimer.
  • Novagen ProteoJuice P/P forms non-covalent complexes with reagent, which differs substantially from protein delivery systems based on lipids or protein transduction domains.
  • Polyplus PULsin P/P forms non-covalent transfections complexes with cationic amphiphile molecule.
  • P/P transfection agents can be classified into three major groups: i) cationic peptides; ii) cationic lipids; iii) other cationic reagents/polymers (e.g. polyamines).
  • transfection agents can be covalently bound to target peptide/protein: by i) genetic fusion (e.g. cationic peptides); ii) by chemical conjugation.
  • the present invention provides a composition for delivering a peptide or protein into a cell, which composition comprises a polycationic agent and a polyanionic agent, wherein the polyanionic agent comprises an inorganic polyphosphate or a polyoxometalate.
  • kit for delivering a peptide or protein into a cell which kit comprises a polycationic agent in a first container and a polyanionic agent in a second container, wherein the polyanionic agent comprises an inorganic polyphosphate or a polyoxometalate.
  • the present invention provides use of a composition for delivering a peptide or protein into a target cell, which composition comprises a polycationic agent and a polyanionic agent, wherein:
  • kit for delivering a peptide or protein into a target cell which kit comprises a polycationic agent in a first container and a polyanionic agent in a second container, wherein;
  • compositions comprising a polycationic agent and a polyanionic agent in which the polyanionic agent comprises a polyphosphate or a polyoxometalate has improved properties for delivering a peptide or protein into a cell.
  • Peptide or protein delivery according to the invention is characterised by a more efficient, uniform and robust performance over a wide range of peptides and proteins of interest, including those of different pI and those of different molecular mass. When compared with prior art methods, it was found that transfection efficiency according to the invention was increased.
  • positively charged cationic agents alone are capable of interacting efficiently with negatively charged peptides or proteins. They are thought to be condensed into nano-sized complexes termed polyplexes which deliver encased peptide or protein material into the cell. Whilst such transfection reagents as in the prior art may be capable of delivering negatively charged biomolecules such as nucleic acids and proteins, different proteins have neutral, negative or positive charges and therefore are not thought to interact with the cationic agents with the same efficiency. According to the invention, by adding a polyanionic agent, its interaction with the peptide or protein of interest may assist in overcoming this problem and ensure that the polyanionic agent coated protein will have a negative charge.
  • the polyanionic and polycationic agents of the invention interact with each other ionically. They may also interact ionically with the peptide or protein of interest. This is an advantageous property because there is no need, for example, for the polyanionic agent to have a functional group for covalent coupling to the peptide or protein. Likewise, there is no need for the polycationic agent to have such a functional group. Accordingly, the composition does not need to incorporate an agent for covalent coupling to the peptide or protein.
  • no agent for covalent coupling is present in the composition.
  • the polycationic agent of the invention may be lipidic, for example comprising a plurality of cationic lipids which may be organised in a supramolecular structure such as a liposome.
  • a polycationic agent which comprises a cationic polymer is preferred.
  • Such cationic polymers may be polylysine or polyarginine,
  • Particularly suitable cationic polymers include polyalkyleneimine polymers such as polyethyleneimines (such as described in U.S. Pat. No. 6,013,240) or polyhydroxyalkyleneimines such as those described in EP2070970A, the contents of which are hereby incorporated by reference.
  • Cationic polyalkyleneimine polymers having a repeat unit comprising a hydrophilic group are described in EP2070970A in which the hydrophilic group is in the polymer backbone or pendant from the polymer backbone and wherein the hydrophilic group preferably comprises a heteroatom.
  • Polyhydroxyalkyleneimine polymers are preferred, such as poly(2-hydroxy)propyleneimine.
  • the polyanionic agent is preferably an anionic polymer, which may be an organic or inorganic polymer.
  • the anionic polymer comprises a polyphosphate, which may be a heterophosphate or a homophosphate.
  • the heterophosphate may comprise a nucleic acid in the form of an oligo or polynucleotide such as DNA or a synthetic analogue thereof, GNA, TNA and LNA being typical examples. It is preferred that the polycationic agent is not lipidic, especially where a nucleic acid is used as the polyanionic agent according to the invention.
  • polyphosphate comprises a homophosphate
  • this may be an inorganic phosphate such as sodium polyphosphate (NaPO 3 ) x .
  • the anionic polymer may comprise a polyoxometalate.
  • Polyoxometalates are polyatomic ions, usually anions, which comprise three or more transition metal oxyanions linked together with shared oxygen atoms to form large, closed 3-dimensional frameworks.
  • the metal atoms that make up the frameworks are sometimes called addenda atoms and the framework may comprise one or more different addenda atoms. These addenda atoms are typically group 5 or group 6 transition metals and may be present in the framework in high oxidation states. Examples of the transition metal atoms include molybdenum and tungsten.
  • the framework of the polyoxometalates may optionally incorporate one or more heteroatoms such as phosphorus. As discussed in further detail below, ammonium molybdate tetrahydrate, sodium phosphotungstate tribasic hydrate and sodium phosphomolybdate hydrate are all useful in the present invention, especially sodium polytungstate.
  • compositions according to the invention may be used to deliver a wide range of peptides or proteins as discussed herein.
  • peptides or proteins include enzymes such as ⁇ -galactosidase, antibodies and inert proteins such as bovine serum albumin. It is possible according to the invention to deliver polypeptides of different pIs and different molecular masses, as discussed further in the examples set out below.
  • the invention may be used to deliver peptides or proteins to a variety of cells, including cells in suspension, adherent cells and primary cells.
  • the invention may be used to deliver to cells in vitro or in vivo.
  • Kits according to the invention may be supplied with additional containers containing suitable buffers or other reagents for use with the polyanionic and polycationic agents according to the invention. Instructions for use of the kit may also be supplied therewith.
  • the present invention provides a method for delivering a peptide or protein into a target cell, which method comprises contacting the peptide or protein with a polycationic agent and a polyanionic agent to form a complex and contacting the complex with the target cell so as to deliver the peptide or protein thereto, wherein;
  • the polyanionic agent it is possible to contact the polyanionic agent with the polycationic agent and with the peptide or protein in any order.
  • the polyanionic agent is contacted with the peptide or protein prior to contact with the polycationic agent. It is thought that the peptide or protein may form a complex with the polyanionic agent and this complex then interacts with the polycationic agent to form a delivery complex.
  • Complexes according to the invention are advantageously formed by ionic interactions.
  • the invention it is possible to deliver the peptides or proteins to cells in vitro, for example in a molecular biology application such as in the study of the role of a protein in the regulation of different cellular processes.
  • compositions of the invention may be used for therapeutic or diagnostic applications as for example in the manufacture of a medicament.
  • a therapeutic protein or peptide may be delivered to a subject in vivo.
  • compositions for the manufacture of a medicament for delivering a peptide or protein into a cell which composition comprises a polycationic agent and a polyanionic agent, wherein;
  • the medicament acts a vehicle for delivering the therapeutic peptide or protein and would need to be prepared to a level of purity and stability suitable for administration to a subject.
  • a product comprising a peptide or protein, a polycationic agent, and a polyanionic agent as a combined preparation for use in medicine, wherein;
  • the combined preparation of the invention typically comprises either a kit for delivering a therapeutic peptide or therapeutic protein in which the peptide or protein, polyanionic agent and polycationic agent are stored in separate containers; or a unitary composition in which each component is present so as to form a complex for administration to the subject.
  • FIG. 1 shows a comparison between embodiments of the invention and the prior art as requested by transfection efficiency and mean fluorescence intensity when delivering labelled antibody to HeLa cells;
  • FIG. 2 shows a comparison between embodiments of the invention and the prior art as measured by distribution of transduced ⁇ -galactosidase in cytoplasmic and membrane protein fractions;
  • FIG. 3 shows a comparison between different polyanions in relation to the enhancement of cationic polymer mediated protein transfection as measured by transfection efficiency and mean fluorescence intensity;
  • FIG. 4 shows a comparison between different polyanions in the enhancement of protein transfection efficiency by polycations pHP and LPEI, as measured by transfection efficiency and mean fluorescence intensity;
  • FIG. 5 a shows polyanion enhancement of pHP-mediated peptide and inert protein transfection as measured by transfection efficiency and mean fluorescence intensity
  • FIG. 5 b shows the enhancement of transfection by polyanion PWp of proteins having different pI as measured by transfection efficiency and mean fluorescence intensity
  • FIG. 6 shows polyanion enhancement of pHP-mediated protein transfection into primary and suspension cells as measured by transfection efficiency, toxicity and mean fluorescence intensity
  • FIG. 7 shows the effect of component mixing on protein transfection as measured by transfection efficiency, toxicity and mean fluorescence intensity.
  • Transfection efficiency of Polyhydroxypropyleneimine (pHP) was initially tested on HeLa cells using Alexa Fluor (AF) 488-labeled antibody (goat IgG) as a control protein.
  • AF Alexa Fluor
  • Different polyanions DNA, sodium polyphosphate (pP) and sodium polytungstate (pW) were tested as additives aiming to improve complex formation.
  • Commercial protein transfection reagents Chariot (Ambion) and ProJect (Pierce) were used as positive controls. The transfection efficiency was evaluated using three criteria: the percent of AF488 positive cells, the percent of dead cells (toxicity) and the mean fluorescence intensity (MFI).
  • the polytungstate evidently is more effective and mediates the biggest amount of protein (MFI ⁇ 370) being delivered into the cell, which is significantly higher than that shown for Chariot or ProJect (MFI ⁇ 100 and 150, respectively).
  • the cells were transfected with ⁇ -galactosidase using the same compositions and protocols as described above.
  • the cells were further fractionated using ProteoJETTM Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytosolic proteins. Enzymatic activity of ⁇ -galactosidase was estimated in both fractions ( FIG. 2 ). Results show that majority of ⁇ -gal activity was detected in the cytosolic fraction for all pHP and ProJect-mediated transfections, while very little or no ⁇ -gal activity was detected in the membrane fraction of pHP-transfected cells, suggesting that cationic polymer (with or without polyanions) positions transduced proteins exclusively inside the cell.
  • polyanion-polycation combination enhances the transfection of proteins with (i) different size, (ii) different pI and (iii) different function.
  • HeLa Human cervical carcinoma-derived cell line
  • Alexa Fluor 488-labeled goat IgG antibody (1 ⁇ g) was diluted in 100 ⁇ l of 0.15M NaCl solution and mixed with different amounts of polyanions: DNA (1 ⁇ g pUC18), sodium polyphosphate (10 mM pP-1 ⁇ l) or sodium polytungstate (10 Mm pW-3 ⁇ l). Cationic polymer pHP (1 ⁇ l) was added into the protein-polyanion mixture and vortexed immediately for few seconds to ensure even distribution of the material. The complexes were allowed to form for 15-20 min at room temperature and added to the cell culture in a drop-wise manner. The cells were further incubated for 2 h at 37° C. in a CO 2 incubator.
  • HeLa cell transfection with ⁇ -galactosidase (1 ⁇ g) was carried out using the same protocol and conditions as described above.
  • the cells were further fractionated using ProteoJETTM Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytoplasmic proteins.
  • the enzymatic activity of ⁇ -galactosidase in both fractions was estimated using colorimetric assay.
  • Polyanions were tested for their ability to improve labelled-IgG transfection.
  • Polyanions were grouped as follows: (i) phosphates—heterophosphates (DNA) and homophosphates (sodium polyphosphate—pP), (ii) POMs—without hetero atom (sodium polytungstate, pW, or ammonium molybdate tetrahydrate, pMo) and with hetero atom (sodium phosphotungstate tribasic hydrate, pWP, and sodium phosphomolybdate hydrate, pMoP).
  • HeLa cells were prepared for transfection essentially as described in Example 1.
  • each polyanion used was: 3 ⁇ l of pW, pWP or pMoP, and 2 ⁇ l of pMo (each 10 mM stock concentration), 0.5 ⁇ l of pP (30 mM stock concentration), 1 ⁇ g of DNA.
  • Polyanion-Antibody-pHP mixtures were incubated for 15 min and added to the cells in a drop-wise manner. Transfection results were processed 2 h later using Guava Easy Cyte Plus flow cytometry system (Millipore).
  • Cationic polymer LPEI (ExGen 500) was tested in Alexa Fluor 488-labeled goat IgG transfection using polyanions—DNA, pP and pW to assist the protein packaging prior to complexation with LPEI.
  • Chinese hamster ovary cells (CHOk1) were prepared for transfection essentially the same way as HeLa cells (example 1). The cells were cultured in RPMI medium supplemented with 10% FBS, the transfection was carried out in serum free RPMI medium. The complexes were formed the same way as described in example 1 for pHP, the amount of LPEI used—3.3 ⁇ l.
  • FITC-labeled proteins 5 kDa peptide, BSA, amyloglucosidase (pI 3.5), ⁇ -lactoglobulin (pI 5.5) and cytochrome-C (pI 10.5) were transfected into HeLa cells following the procedure described in Example 1.
  • HeLa cells were prepared for transfection as described in Example 1.
  • the antibody IgG (1 ⁇ g)-pWP (1 ⁇ l)-pHP (1 ⁇ l) complexes were prepared in 0.15 M NaCl following different component mixing order: IgG+PA+pHP, PA+IgG+pHP, IgG+pHP+PA, pHP+IgG+PA PA+pHP+IgG, and pHP+PA+IgG.
  • Complexes were allowed to form for 15 min and added to the cells in a drop-wise manner.

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Abstract

A composition and method comprising a polycationic agent and a polyanionic agent for delivering a peptide or protein into a cell.

Description

This application claims priority from co-pending GB Application No. 0917792.4 filed Oct. 12, 2009.
FIELD OF INVENTION
The present invention relates to a composition for delivering a peptide or protein into a cell, a kit for delivering a peptide or protein into a cell and a method therefor, as well as uses of the compositions and kits.
BACKGROUND OF INVENTION Abbreviations
  • P/P—peptide or protein
  • PA—polyanionic agent
  • pHP—polyhydroxypropyleneimine
  • pWP—sodium phosphotungstate tribasic hydrate (Na3O40PW12.xH2O);
  • pW—sodium polytungstate (Na6O39W12.H2O);
  • pMoP—sodium phosphomolybdate hydrate (Mo12Na3O40P.xH2O);
  • pMo—ammonium molybdate tetrahydrate (H24M7N6O24.4H2O);
  • pP—sodium polyphosphate (NaPO3)12-13xNa2O
Efficient foreign protein delivery into living cells can completely bypass transcription-translation processes related to gene expression, reducing the time required for target protein synthesis from days to hours.
The ideal peptide/protein (P/P) delivery method/agent into eukaryotic cells should meet three major criteria: i) it should efficiently bring P/P into the cells; ii) it should protect P/P against denaturation and degradation; iii) the method itself should be non-toxic to the host cells.
In general, P/P delivery or transfection techniques include physical delivery methods, such as microinjection, electroporation, and methods based on chemical transfection agents of different origin that facilitate protein delivery into the cell. A general characteristic of a protein delivery agent is its ability to form positively charged complexes with target P/P, which are capable of attaching themselves to negatively charged cell surfaces, crossing through the plasma membrane and delivering the captured protein into the cell. Usually the transfection agent stabilizes the protein, protects it from degradation and preserves its natural characteristics during transfection.
The most popular commercially available current protein delivery agents are presented in Table 1.
TABLE 1
Peptide/protein (P/P) delivery into cells.
Company Product Characteristics Method/agent reference
Active Motif Chariot P/P forms non-covalent U.S. Pat. No. 6,841,535
complex with Chariot
peptide
GenScript Pep-1 P/P forms non-covalent U.S. Pat. No. 6,841,535
Corp. complex with Pep-1
peptide
Invitrogen Voyager Voyager products are U.S. Pat. No. 6,184,038
bacterial and mammalian
expression systems for
the production of
polypeptides fused with
VP22.
Krackeler TransVector TransVector is a U.S. Pat. No. 6,080,724
Scientific bacterial expression
Inc./ system for the production
Qbiogene of purified polypeptides
fused with Penetratin.
Krackeler Penetratin 1 peptide Penetratin 1 peptide is U.S. Pat. No. 6,080,724
Scientific activated peptide for
Inc./ chemical conjugation
Qbiogene with target P/P.
Panomics DeliverX P/P forms non-covalent U.S. Pat. No. 6,841,535
nanoparticles with virus-
derived amphipathic
peptides (MPG peptide
technology)
Imgenex ProVectin Encapsulation of P/P in
liposomes or
association of P/P with
liposomal membrane.
Genlantis/ BioPorter P/P forms non-covalent WO03095641A1
Gene Therapy complexes with
Systems cationic lipids mixture.
Pierce Pro-Ject P/P forms non-covalent
complexes with
cationic lipids mixture.
Stratagene BioTrek P/P forms non-covalent
complexes with lipid
formulated reagent.
Synvolux SAINT PhD P/P forms non-covalent EP0755924B1
Therapeutics complexes with
cationic pyridinium
amphiphile and a helper
lipid.
Targeting ProFect-P1, P/P forms non-covalent
Systems ProFect-P2 complexes with: i) lipid
reagent (ProFect-P1);
ii) with non-lipid
reagent (ProFect-P2).
NEB TransPass P P/P forms non-covalent
complexes with non-lipid
polimer.
Novagen ProteoJuice P/P forms non-covalent
complexes with reagent,
which differs
substantially from
protein delivery systems
based on lipids or protein
transduction domains.
Polyplus PULsin P/P forms non-covalent
transfections complexes with cationic
amphiphile molecule.
Based on the origin of material, P/P transfection agents can be classified into three major groups: i) cationic peptides; ii) cationic lipids; iii) other cationic reagents/polymers (e.g. polyamines). As an alternative, transfection agents can be covalently bound to target peptide/protein: by i) genetic fusion (e.g. cationic peptides); ii) by chemical conjugation. There are many known peptide/protein delivery methods that have been successfully commercialized, however, all of them have certain limitations in their application or efficacy regarding the type of target protein, its pI or molecular weight or the types of cells used for transfection. Therefore, there is a need in the market for efficient, universal and robust protein delivery agents.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a composition for delivering a peptide or protein into a cell, which composition comprises a polycationic agent and a polyanionic agent, wherein the polyanionic agent comprises an inorganic polyphosphate or a polyoxometalate.
There is further provided a kit for delivering a peptide or protein into a cell, which kit comprises a polycationic agent in a first container and a polyanionic agent in a second container, wherein the polyanionic agent comprises an inorganic polyphosphate or a polyoxometalate.
In a further aspect, the present invention provides use of a composition for delivering a peptide or protein into a target cell, which composition comprises a polycationic agent and a polyanionic agent, wherein:
    • (1) the polyanionic agent comprises a polyphosphate or a polyoxometalate; and/or
    • (2) the polycationic agent comprises a polyalkyleneimine.
There is further provided use of a kit for delivering a peptide or protein into a target cell, which kit comprises a polycationic agent in a first container and a polyanionic agent in a second container, wherein;
    • (1) the polyanionic agent comprises a polyphosphate or a polyoxometalate; and/or
    • (2) the polycationic agent comprises a polyalkyleneimine.
It has surprisingly been found that a composition comprising a polycationic agent and a polyanionic agent in which the polyanionic agent comprises a polyphosphate or a polyoxometalate has improved properties for delivering a peptide or protein into a cell. Peptide or protein delivery according to the invention is characterised by a more efficient, uniform and robust performance over a wide range of peptides and proteins of interest, including those of different pI and those of different molecular mass. When compared with prior art methods, it was found that transfection efficiency according to the invention was increased.
Without wishing to be bound by theory, it is thought that positively charged cationic agents alone are capable of interacting efficiently with negatively charged peptides or proteins. They are thought to be condensed into nano-sized complexes termed polyplexes which deliver encased peptide or protein material into the cell. Whilst such transfection reagents as in the prior art may be capable of delivering negatively charged biomolecules such as nucleic acids and proteins, different proteins have neutral, negative or positive charges and therefore are not thought to interact with the cationic agents with the same efficiency. According to the invention, by adding a polyanionic agent, its interaction with the peptide or protein of interest may assist in overcoming this problem and ensure that the polyanionic agent coated protein will have a negative charge. This enables it to interact efficiently with the polycationic agent and subsequently be transported through the cell membrane. This is thought to give rise to delivery agents which are more efficient, and which offer a more uniform and robust performance over a wide range of peptides or proteins of interest rather than just some.
Typically, the polyanionic and polycationic agents of the invention interact with each other ionically. They may also interact ionically with the peptide or protein of interest. This is an advantageous property because there is no need, for example, for the polyanionic agent to have a functional group for covalent coupling to the peptide or protein. Likewise, there is no need for the polycationic agent to have such a functional group. Accordingly, the composition does not need to incorporate an agent for covalent coupling to the peptide or protein. Advantageously, no agent for covalent coupling is present in the composition.
The polycationic agent of the invention may be lipidic, for example comprising a plurality of cationic lipids which may be organised in a supramolecular structure such as a liposome. A polycationic agent which comprises a cationic polymer is preferred. Such cationic polymers may be polylysine or polyarginine, Particularly suitable cationic polymers include polyalkyleneimine polymers such as polyethyleneimines (such as described in U.S. Pat. No. 6,013,240) or polyhydroxyalkyleneimines such as those described in EP2070970A, the contents of which are hereby incorporated by reference. Cationic polyalkyleneimine polymers having a repeat unit comprising a hydrophilic group are described in EP2070970A in which the hydrophilic group is in the polymer backbone or pendant from the polymer backbone and wherein the hydrophilic group preferably comprises a heteroatom. Polyhydroxyalkyleneimine polymers are preferred, such as poly(2-hydroxy)propyleneimine.
The polyanionic agent is preferably an anionic polymer, which may be an organic or inorganic polymer. In one arrangement the anionic polymer comprises a polyphosphate, which may be a heterophosphate or a homophosphate. The heterophosphate may comprise a nucleic acid in the form of an oligo or polynucleotide such as DNA or a synthetic analogue thereof, GNA, TNA and LNA being typical examples. It is preferred that the polycationic agent is not lipidic, especially where a nucleic acid is used as the polyanionic agent according to the invention.
Where the polyphosphate comprises a homophosphate, this may be an inorganic phosphate such as sodium polyphosphate (NaPO3)x.
Alternatively, the anionic polymer may comprise a polyoxometalate. Polyoxometalates are polyatomic ions, usually anions, which comprise three or more transition metal oxyanions linked together with shared oxygen atoms to form large, closed 3-dimensional frameworks. The metal atoms that make up the frameworks are sometimes called addenda atoms and the framework may comprise one or more different addenda atoms. These addenda atoms are typically group 5 or group 6 transition metals and may be present in the framework in high oxidation states. Examples of the transition metal atoms include molybdenum and tungsten. The framework of the polyoxometalates may optionally incorporate one or more heteroatoms such as phosphorus. As discussed in further detail below, ammonium molybdate tetrahydrate, sodium phosphotungstate tribasic hydrate and sodium phosphomolybdate hydrate are all useful in the present invention, especially sodium polytungstate.
Compositions according to the invention may be used to deliver a wide range of peptides or proteins as discussed herein. Such peptides or proteins include enzymes such as β-galactosidase, antibodies and inert proteins such as bovine serum albumin. It is possible according to the invention to deliver polypeptides of different pIs and different molecular masses, as discussed further in the examples set out below.
The invention may be used to deliver peptides or proteins to a variety of cells, including cells in suspension, adherent cells and primary cells. The invention may be used to deliver to cells in vitro or in vivo.
Kits according to the invention may be supplied with additional containers containing suitable buffers or other reagents for use with the polyanionic and polycationic agents according to the invention. Instructions for use of the kit may also be supplied therewith.
In a further aspect the present invention provides a method for delivering a peptide or protein into a target cell, which method comprises contacting the peptide or protein with a polycationic agent and a polyanionic agent to form a complex and contacting the complex with the target cell so as to deliver the peptide or protein thereto, wherein;
    • (1) the polyanionic agent comprises a polyphosphate or a polyoxometalate; and/or
    • (2) the polycationic agent comprises a polyalkyleneimine.
According to the method of the invention, it is possible to contact the polyanionic agent with the polycationic agent and with the peptide or protein in any order. In a preferred embodiment, the polyanionic agent is contacted with the peptide or protein prior to contact with the polycationic agent. It is thought that the peptide or protein may form a complex with the polyanionic agent and this complex then interacts with the polycationic agent to form a delivery complex. Complexes according to the invention are advantageously formed by ionic interactions.
According to the invention it is possible to deliver the peptides or proteins to cells in vitro, for example in a molecular biology application such as in the study of the role of a protein in the regulation of different cellular processes.
In a further aspect the compositions of the invention may be used for therapeutic or diagnostic applications as for example in the manufacture of a medicament. According to this aspect, for example, a therapeutic protein or peptide may be delivered to a subject in vivo.
Thus, there is further provided use of a composition for the manufacture of a medicament for delivering a peptide or protein into a cell, which composition comprises a polycationic agent and a polyanionic agent, wherein;
    • (1) the polyanionic agent comprises a polyphosphate or a polyoxometalate; and/or
    • (2) the polycationic agent comprises a polyalkyleneimine.
In this way, the medicament acts a vehicle for delivering the therapeutic peptide or protein and would need to be prepared to a level of purity and stability suitable for administration to a subject.
There is further provided a product comprising a peptide or protein, a polycationic agent, and a polyanionic agent as a combined preparation for use in medicine, wherein;
    • (1) the polyanionic agent comprises a polyphosphate or a polyoxometalate; and/or
    • (2) the polycationic agent comprises a polyalkyleneimine.
In this further aspect, the combined preparation of the invention typically comprises either a kit for delivering a therapeutic peptide or therapeutic protein in which the peptide or protein, polyanionic agent and polycationic agent are stored in separate containers; or a unitary composition in which each component is present so as to form a complex for administration to the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in further detail, by way of example only, with reference to the accompanying drawings in which:
FIG. 1 shows a comparison between embodiments of the invention and the prior art as requested by transfection efficiency and mean fluorescence intensity when delivering labelled antibody to HeLa cells;
FIG. 2 shows a comparison between embodiments of the invention and the prior art as measured by distribution of transduced β-galactosidase in cytoplasmic and membrane protein fractions;
FIG. 3 shows a comparison between different polyanions in relation to the enhancement of cationic polymer mediated protein transfection as measured by transfection efficiency and mean fluorescence intensity;
FIG. 4 shows a comparison between different polyanions in the enhancement of protein transfection efficiency by polycations pHP and LPEI, as measured by transfection efficiency and mean fluorescence intensity;
FIG. 5 a shows polyanion enhancement of pHP-mediated peptide and inert protein transfection as measured by transfection efficiency and mean fluorescence intensity;
FIG. 5 b shows the enhancement of transfection by polyanion PWp of proteins having different pI as measured by transfection efficiency and mean fluorescence intensity;
FIG. 6 shows polyanion enhancement of pHP-mediated protein transfection into primary and suspension cells as measured by transfection efficiency, toxicity and mean fluorescence intensity; and
FIG. 7 shows the effect of component mixing on protein transfection as measured by transfection efficiency, toxicity and mean fluorescence intensity.
 DETAILED DESCRIPTION OF THE INVENTION
Transfection efficiency of Polyhydroxypropyleneimine (pHP) was initially tested on HeLa cells using Alexa Fluor (AF) 488-labeled antibody (goat IgG) as a control protein. Different polyanions: DNA, sodium polyphosphate (pP) and sodium polytungstate (pW) were tested as additives aiming to improve complex formation. Commercial protein transfection reagents Chariot (Ambion) and ProJect (Pierce) were used as positive controls. The transfection efficiency was evaluated using three criteria: the percent of AF488 positive cells, the percent of dead cells (toxicity) and the mean fluorescence intensity (MFI).
The results show that antibody cannot internalize into the cell on its own (FIG. 1). The amount of AF488-positive cells increased to 65% when cationic polymer pHP was used in complex with the antibody. The percent of transfected cells was even higher (up to 85-95%) when different polyanions (DNA, pP or pW) were added into the mixture, suggesting that polyanions have positive effect for protein delivery. Polyanions alone, on the other hand, have no effect on protein entry into the cell (FIG. 3). Comparison of obtained antibody transfection results with two commonly used commercial protein transfection reagents—Chariot and ProJect, reveals very similar transfection efficiencies (85% for Chariot and 80% for ProJect). However, when comparing the MFI values, the polytungstate evidently is more effective and mediates the biggest amount of protein (MFI˜370) being delivered into the cell, which is significantly higher than that shown for Chariot or ProJect (MFI˜100 and 150, respectively).
To determine cellular localization of transduced proteins, the cells were transfected with β-galactosidase using the same compositions and protocols as described above. The cells were further fractionated using ProteoJET™ Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytosolic proteins. Enzymatic activity of β-galactosidase was estimated in both fractions (FIG. 2). Results show that majority of β-gal activity was detected in the cytosolic fraction for all pHP and ProJect-mediated transfections, while very little or no β-gal activity was detected in the membrane fraction of pHP-transfected cells, suggesting that cationic polymer (with or without polyanions) positions transduced proteins exclusively inside the cell. For ProJect, however, considerable amount of β-gal activity was found in the membrane, indicating that equivalent amount of protein after transfection remains stuck within or on the surface of the cellular membrane. For Chariot-mediated transfection, significantly more β-galactosidase was found in the membrane fraction than in the cytosol. In conclusion, pHP-polyanion mixture facilitates highly efficient protein transduction resulting primarily in cytosolic protein localization inside the cell.
To examine if other polyanions contribute to cationic polymer-mediated protein transduction, we tested sodium phosphomolybdate hydrate (pMoP), ammonium molybdate tetrahydrate (pMo), as well as sodium phosphotungstate tribasic hydrate (pWP) along with previously used polyanions: DNA, pP and pW (FIG. 3). The results show that all analyzed polyanions enhance protein transfection to a similar level of 80-95%. The MFI data, however, singled out polytungstates (with or without hetero atoms) as the most potent enhancers (MFI˜1200). The polyoxometalates (POMs) carrying hetero atom (pWP and pMoP) apparently performed slightly worse than POMs without hetero atom (pW and pMo).
To further investigate if polyanions have positive effect in combination with other polycations used in protein transfections, a popular cationic polymer—LPEI was tested along with pHP in fluorescently labelled antibody transfections. Polyanions—DNA, pP and pW were used to assist protein packaging prior to complexation with LPEI (FIG. 4). The results show that polyanions enhance LPEI-mediated protein transfection as efficiently as pHP-mediated transfection. The MFI values increase from 10 units (protein-LPEI) to 25, 60 and 130 units upon addition of DNA, pP or pW, respectively. The results suggest that negatively charged polyanions may interact with positively charged regions of the antibody and consequently facilitate protein-polyanion interaction with the positively charged polycation.
To demonstrate that polyanions are able to enhance transfection of any type protein, a number of proteins of different size, pI value or function were chemically conjugated to FITC and examined using the same conditions as those used for antibody transfections described above. Successful delivery of 5 kDa peptide (FIG. 5 a), 12 kDa cytochrome C, 18 kDa β-lactoglobulin (FIG. 5 b), 66 kDa BSA, 97 kDa amyloglucosidase, as well as earlier tested 116 kDa β-galactosidase (FIG. 2) and 150 kDa IgG (FIG. 1, 3, 4), confirm that polyanions enhance transfection of any size protein carried by cationic polymer pHP. The MFI values primarily depend on the size of the protein, i.e. the extent of FITC labelling. Smaller proteins had lower number of FITC molecules and, as a result, their fluorescence was weaker. The transfection of proteins bearing different pI (amyloglucosidase—pI 3.5, β-lactoglobulin pI 5.5, cytochrome C pI 10.5) gave similar results (FIG. 5 b), all three proteins were delivered with ˜90% efficiency. The amount of polyanion used in this case, depended on the pI of the protein: less polyanion was needed for transfection of amyloglucosidase (0.5 μl), more for transfection of cytochrome C (1.0 μl). Overall, the results show, that polyanion-polycation combination enhances the transfection of proteins with (i) different size, (ii) different pI and (iii) different function.
For the final evaluation of polyanion exerted effect on the pHP-mediated protein transfection the experiments were carried on different cell types: primary human lung fibroblasts (primary cells—usually difficult to transfect), HeLa S3 (loosely adherent cell line), and Jurkat T cell lymphoma cells (suspension cell line—known to be very difficult to transfect by chemical methods). The results showed that irrespective of the cell type used, the transfection efficiencies reached 90% (FIG. 6). The fluorescence level in strongly adherent HLF cells was the highest (MFI˜1500), while in semiadherent or suspension cells, HeLa S3 and Jurkat, the MFI was 350 and 500, respectively, suggesting that the extent of macromolecule uptake depends on the cell type. Cell size in this experiment should be taken into consideration as well, since HLF cells are significantly bigger, can internalize more material than HeLa S3 or Jurkat cells, and thus fluoresce more intensively than smaller cells. In conclusion, the polyanion (here, pWP) grouping with protein prior to complexation with polycation (pHP) facilitates efficient protein delivery into the primary, adherent and suspension cell types.
Evaluation of polyanion-protein-polycation complex formation after different component mixing schedule and its influence on transfection efficiency was carried out in order to determine the best possible way to form protein-pHP complexes and to ensure the most efficient cargo transport through the cellular membrane. The results apparently were very similar, no matter how the components were mixed together (FIG. 7), the transfection efficiencies ranged from 80 to 95%. Slightly lower MFI values (200 units) suggested that protein should not be the last element added into the mix, but rather mixed with either polycation or polyanion first.
Example 1 Analysis of the Protein Transfection Using pHP
Transfection of HeLa (Human cervical carcinoma-derived cell line) cells was carried out as follows: one day before the transfection experiment, the cells were seeded in a 24-well tissue culture plate at the density of 5×104 cells per well in the total volume of 1 ml DMEM culture medium supplemented with 10% FBS. The cells were incubated at 37° C. in a CO2 incubator until they reached 70-80% confluency (usually within 24 h). On the day of transfection, the growth medium was removed and replaced with 0.5 ml of warm serum-free medium. Alexa Fluor 488-labeled goat IgG antibody (1 μg) was diluted in 100 μl of 0.15M NaCl solution and mixed with different amounts of polyanions: DNA (1 μg pUC18), sodium polyphosphate (10 mM pP-1 μl) or sodium polytungstate (10 Mm pW-3 μl). Cationic polymer pHP (1 μl) was added into the protein-polyanion mixture and vortexed immediately for few seconds to ensure even distribution of the material. The complexes were allowed to form for 15-20 min at room temperature and added to the cell culture in a drop-wise manner. The cells were further incubated for 2 h at 37° C. in a CO2 incubator. To remove unincorporated complexes, the cultures were rinsed with PBS, and the cells were analyzed by FACS (Fluorescence Activated Cell Sorter). Transfections using Chariot (Ambion) and ProJect (Pierce) reagents were carried out following manufacturer suggested protocols.
Example 2 Analysis of the Protein Localization after Polyanion-Polycation Mediated Transfection
HeLa cell transfection with β-galactosidase (1 μg) was carried out using the same protocol and conditions as described above. The cells were further fractionated using ProteoJET™ Membrane Protein Extraction Kit (Fermentas) in order to separate membrane and cytoplasmic proteins. The enzymatic activity of β-galactosidase in both fractions was estimated using colorimetric assay.
Example 3 Analysis of Different Polyanions in pHP-Mediated Protein Transfections
Several different polyanions were tested for their ability to improve labelled-IgG transfection. Polyanions were grouped as follows: (i) phosphates—heterophosphates (DNA) and homophosphates (sodium polyphosphate—pP), (ii) POMs—without hetero atom (sodium polytungstate, pW, or ammonium molybdate tetrahydrate, pMo) and with hetero atom (sodium phosphotungstate tribasic hydrate, pWP, and sodium phosphomolybdate hydrate, pMoP). HeLa cells were prepared for transfection essentially as described in Example 1. The amount of each polyanion used was: 3 μl of pW, pWP or pMoP, and 2 μl of pMo (each 10 mM stock concentration), 0.5 μl of pP (30 mM stock concentration), 1 μg of DNA. Polyanion-Antibody-pHP mixtures were incubated for 15 min and added to the cells in a drop-wise manner. Transfection results were processed 2 h later using Guava Easy Cyte Plus flow cytometry system (Millipore).
Example 4 Analysis of Polyanions in Different Polycation-Mediated Protein Transfection
Cationic polymer LPEI (ExGen 500) was tested in Alexa Fluor 488-labeled goat IgG transfection using polyanions—DNA, pP and pW to assist the protein packaging prior to complexation with LPEI. Chinese hamster ovary cells (CHOk1) were prepared for transfection essentially the same way as HeLa cells (example 1). The cells were cultured in RPMI medium supplemented with 10% FBS, the transfection was carried out in serum free RPMI medium. The complexes were formed the same way as described in example 1 for pHP, the amount of LPEI used—3.3 μl.
Example 5 Analysis of Polyanions in Transfections of Proteins of Different Size and pI
FITC-labeled proteins—5 kDa peptide, BSA, amyloglucosidase (pI 3.5), β-lactoglobulin (pI 5.5) and cytochrome-C (pI 10.5) were transfected into HeLa cells following the procedure described in Example 1. The amount of pWP used: 0.5 μl for amyloglucosidase and β-lactoglobulin, 1 μl for cytochrome C.
Example 6 Analysis of the Polyanion Effect on Difficult to Transfect Cell Lines
Comparison of protein transfer efficiency using pHP and polyanions (pWP) was tested in suspension cell lines, HeLa S3 and Jurkat (Human T cell lymphoma cell line), as well as in primary cells HLF (human lung fibroblasts). Suspension cells were seeded at the density of 2×105 cells/well, HLF 5×104/well 24 hours before the transfection. Antibody-pHP complexes in 0.15 M NaCl solution were prepared as described earlier.
Example 7 Analysis of the Component Mixing Order Effect on Protein Transfection
HeLa cells were prepared for transfection as described in Example 1. The antibody IgG (1 μg)-pWP (1 μl)-pHP (1 μl) complexes were prepared in 0.15 M NaCl following different component mixing order: IgG+PA+pHP, PA+IgG+pHP, IgG+pHP+PA, pHP+IgG+PA PA+pHP+IgG, and pHP+PA+IgG. Complexes were allowed to form for 15 min and added to the cells in a drop-wise manner.

Claims (6)

The invention claimed is:
1. A composition for delivering a peptide or protein into a cell, the composition comprising:
a polycationic delivery agent comprising a polyalkyleneimine or a polyhydroxyalkyleneimine and one of
(a) a polyoxometalate, or
(b) (PO3)x where x is 12-13.
2. The composition according to claim 1, wherein the polyoxometalate comprises a molybdenum or tungsten polyoxometalate and optionally carries a heteroatom.
3. The composition according to claim 1, wherein the polycationic agent comprises a polyhydroxyalkyleneimine.
4. The composition according to claim 3, wherein the polyhydroxyalkyleneimine is polyhydroxypropyleneimine.
5. The composition according to claim 1, wherein the polyalkyleneimine comprises a polyethyleneimine.
6. A pharmaceutical product comprising
a peptide or protein,
a polycationic delivery agent, and
a polyanionic delivery agent
as a combined preparation, where the polyanionic delivery agent comprises
(a) a polyoxometalate, or
(b) (PO3)x where x is 12-13, and
where the polycationic delivery agent comprises a polyalkyleneimine or a polyhydroxyalkyleneimine.
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Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0755924A1 (en) 1995-07-25 1997-01-29 Stichting voor de Technische Wetenschappen Transportvehicles for macromolecules
WO1999036089A1 (en) 1998-01-16 1999-07-22 The Johns Hopkins University Genetic immunization with co-delivery of nucleic acid and cytokines in a single vehicle
US6013240A (en) 1994-07-13 2000-01-11 Rhone-Poulenc Rorer Sa Nucleic acid containing composition, preparation and uses of same
US6080724A (en) 1995-10-05 2000-06-27 Centre National De La Recherche Scientifique (Cnrs) Peptides which can be used as vectors for the intracellular addressing of active molecules
US6184038B1 (en) 1995-07-28 2001-02-06 Marie Curie Cancer Care Transport proteins and their uses
WO2001043778A1 (en) 1999-12-17 2001-06-21 Gene Therapy Systems, Inc. Use of cationic lipids for intracellular protein delivery
US6433017B1 (en) 1998-11-25 2002-08-13 Gene Therapy Systems, Inc. Amphiphilic polyamine compounds
US6566114B1 (en) * 1998-06-10 2003-05-20 Novozymes, A/S Mannanases
EP1316318A2 (en) 2001-11-29 2003-06-04 Nippon Shokubai Co., Ltd. Method of transducing a protein into cells
WO2003095641A1 (en) 2002-05-06 2003-11-20 Gene Therapy Systems, Inc. Intracellular protein delivery compositions and methods of use
US6770479B1 (en) 1998-07-10 2004-08-03 The United States Of America As Represented By The Secretary Of The Army Anthrax vaccine
US20040203154A1 (en) 2002-10-04 2004-10-14 Vladimir Trubetskoy Process for generating multilayered particles
WO2004096998A2 (en) 2003-04-29 2004-11-11 Vanderbilt University Nanoparticular tumor targeting and therapy
US6825341B2 (en) 2000-02-18 2004-11-30 Gencell S.A. Process for preparing functionalized polyalkyleneimines, compositions containing them and uses thereof
US6841535B2 (en) 2000-07-31 2005-01-11 Active Motif Peptide-mediated transfection agents and methods of use
US20060204444A1 (en) * 2002-05-17 2006-09-14 Montana State University National Institute Of Health Protein cages for the delivery of medical imaging and therapeutic agents
EP2070970A2 (en) 2007-12-12 2009-06-17 Fermentas UAB Transfection Reagent
WO2009142893A2 (en) 2008-05-19 2009-11-26 The University Of North Carolina At Chapel Hill Methods and compositions for the delivery of bioactive compounds
US8173131B2 (en) * 2000-10-18 2012-05-08 Perkinelmer Health Sciences, Inc. Compositions and methods for modulating RSV infection and immunity

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013240A (en) 1994-07-13 2000-01-11 Rhone-Poulenc Rorer Sa Nucleic acid containing composition, preparation and uses of same
EP0755924A1 (en) 1995-07-25 1997-01-29 Stichting voor de Technische Wetenschappen Transportvehicles for macromolecules
US6184038B1 (en) 1995-07-28 2001-02-06 Marie Curie Cancer Care Transport proteins and their uses
US6080724A (en) 1995-10-05 2000-06-27 Centre National De La Recherche Scientifique (Cnrs) Peptides which can be used as vectors for the intracellular addressing of active molecules
WO1999036089A1 (en) 1998-01-16 1999-07-22 The Johns Hopkins University Genetic immunization with co-delivery of nucleic acid and cytokines in a single vehicle
US6566114B1 (en) * 1998-06-10 2003-05-20 Novozymes, A/S Mannanases
US6770479B1 (en) 1998-07-10 2004-08-03 The United States Of America As Represented By The Secretary Of The Army Anthrax vaccine
US6433017B1 (en) 1998-11-25 2002-08-13 Gene Therapy Systems, Inc. Amphiphilic polyamine compounds
WO2001043778A1 (en) 1999-12-17 2001-06-21 Gene Therapy Systems, Inc. Use of cationic lipids for intracellular protein delivery
US6825341B2 (en) 2000-02-18 2004-11-30 Gencell S.A. Process for preparing functionalized polyalkyleneimines, compositions containing them and uses thereof
US6841535B2 (en) 2000-07-31 2005-01-11 Active Motif Peptide-mediated transfection agents and methods of use
US8173131B2 (en) * 2000-10-18 2012-05-08 Perkinelmer Health Sciences, Inc. Compositions and methods for modulating RSV infection and immunity
EP1316318A2 (en) 2001-11-29 2003-06-04 Nippon Shokubai Co., Ltd. Method of transducing a protein into cells
WO2003095641A1 (en) 2002-05-06 2003-11-20 Gene Therapy Systems, Inc. Intracellular protein delivery compositions and methods of use
US20060204444A1 (en) * 2002-05-17 2006-09-14 Montana State University National Institute Of Health Protein cages for the delivery of medical imaging and therapeutic agents
US20040203154A1 (en) 2002-10-04 2004-10-14 Vladimir Trubetskoy Process for generating multilayered particles
WO2004096998A2 (en) 2003-04-29 2004-11-11 Vanderbilt University Nanoparticular tumor targeting and therapy
EP2070970A2 (en) 2007-12-12 2009-06-17 Fermentas UAB Transfection Reagent
WO2009142893A2 (en) 2008-05-19 2009-11-26 The University Of North Carolina At Chapel Hill Methods and compositions for the delivery of bioactive compounds

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
Baranauskas, Aurimas, "Biochemical Properties of Methyl-specific Restriction Endonuclease," Biochemistry Master'sThesis, Vilnius University (2006). Abstract only.
BioPorter. Protein Delivery Reagent. http://www.genlantis.com/objects/catalog/product/extras/1077-Resource-Technical-Updates-No6.pdf. Oct. 12, 2009.
BioTrek(TM) Protein Delivery Reagent. Instruction Manual. Catalog #204140. Revision #083001.
BioTrek™ Protein Delivery Reagent. Instruction Manual. Catalog #204140. Revision #083001.
Buyer's Guide to Protein Transduction Reagents. The Scientist. vol. 18, Issue 11, p. 42 (2004).
Chariot(TM) Simple, efficient protein delivery. http://www.activemotif.com/documents/5.pdf. Oct. 12, 2009.
Chariot™ Simple, efficient protein delivery. http://www.activemotif.com/documents/5.pdf. Oct. 12, 2009.
Futami et al., "Intracellular delivery of proteins into mammalian living cells by polyethylenimine cationization", Journal of Bioscience and Bioengineering, vol. 99, No. 2, pp. 95-103, Feb. 2005.
GenScript. Pep-1 (Uncapped). Sep. 29, 2009. http://www.denscript.com/peptide/RP10130-Pep-1-peptide.html. Sep. 29, 2009.
Great Britain Search Report dated Feb. 11, 2010.
Imegenex. ProVectin(TM) Protein Delivery 2009. Reagent. http://www.imgenex.com/provectin.php. Sep. 18, 2009.
Imegenex. ProVectin™ Protein Delivery 2009. Reagent. http://www.imgenex.com/provectin.php. Sep. 18, 2009.
International Search Report and Written Opinion, PCT/EP2010/065236, mailed Mar. 29, 2011.
Invitrogen. Voyager(TM) NES Protein Production Kits. http://www.tools.invitrogen.com/content/sfs/manuals/voyagernesprotprodkits-man.pdf. Apr. 7, 2004.
Invitrogen. Voyager™ NES Protein Production Kits. http://www.tools.invitrogen.com/content/sfs/manuals/voyagernesprotprodkits—man.pdf. Apr. 7, 2004.
Jay Harlow (2002) "Sea Scallops: How to Avoid Getting Soaked", published on Sally's Place, online, http://www.sallybernstein.com/food/columns/harlow/sea-scallops.htm, no journal, volume, number of pages, 4 pages long in printout. *
Klima{hacek over (s)}auskas, Saulius, "New enzymes and technologies epigenetics analysis," Project EPIGEN, Biotechnologijos Institutes, Registration No. P-07003, Contract No. P-03/2008 (2008). Abstract only.
Krackeler Scientific, Inc. Penetratin(TM) 1 Peptide. http://www.krackeler.com/products/fid/2819. Sep. 29, 2009.
Krackeler Scientific, Inc. Penetratin™ 1 Peptide. http://www.krackeler.com/products/fid/2819. Sep. 29, 2009.
Krackeler Scientific, Inc. TransVector. http://www.krackeler.com/products/fid/2820. Sep. 29, 2009.
Li et al. Synthesis of lipopolyhydroxyalkyleneamines for gene delivery, Bioorganic & Medicinal Chemistry Letters, 16 (2006), pp. 2428-2432.
Merck ProteoJuice(TM)Protein Transfection Reagent. http://www.merckbiosciences.co.uk/Products/ProductDisplay.asp?catno=71281. Sep. 29, 2009.
Merck ProteoJuice™Protein Transfection Reagent. http://www.merckbiosciences.co.uk/Products/ProductDisplay.asp?catno=71281. Sep. 29, 2009.
New England BioLabs Inc. TransPass(TM) P Protein Transfection Reagent. http://www.neb.com/nebecomm/products/productM2563.asp. Sep. 29, 2009.
New England BioLabs Inc. TransPass™ P Protein Transfection Reagent. http://www.neb.com/nebecomm/products/productM2563.asp. Sep. 29, 2009.
Panomics. DeliverX(TM) Peptide Transfection Kit Product Information. Copyright date 2006.
Panomics. DeliverX(TM) Peptide Transfection Kits. http://www.panomics.com/product/43/. Sep. 29, 2009.
Panomics. DeliverX™ Peptide Transfection Kit Product Information. Copyright date 2006.
Panomics. DeliverX™ Peptide Transfection Kits. http://www.panomics.com/product/43/. Sep. 29, 2009.
PolyPlus Transfection. PULSin(TM) Delivery Reagent of Functional Proteins/Antibodies and Peptides to Living Cells. http://www.polyplus-transfection.com?EN/produit.php?PAGEID=131. Sep. 18, 2009.
PolyPlus Transfection. PULSin™ Delivery Reagent of Functional Proteins/Antibodies and Peptides to Living Cells. http://www.polyplus-transfection.com?EN/produit.php?PAGEID=131. Sep. 18, 2009.
Profect Protein Delivery Reagents Product Applications Guide 2008. http://www.targetingsystem.net/profect.pdf. Oct. 12, 2009.
Shigeta, et al. (2006) "Anti-RNA Virus Activity of Polyoxometalates", Biomedicine & Pharmacotherapy, 60(5):211-19. *
Synvolux Therapeutics. Protein transfection reagent SAINT-PhD. hhtp://www.synvoluxproducts.com/saintphd.php. Oct. 12, 2009.
Thermo Scientific. Pro-Ject Protein Transfection Reagent. http://www.piercenet.com/Objects/View,cfm?type=productFamily&ID=A21BD675-A083-4D56-A6B0-F1F6BA6DA446. Sep. 18, 2009.
Zaliauskiene et al. Efficient Gene Transfection Using Novel Cationic Polymers Poly(hydroxyalkylene imines). Bioconjugate Chemistry, 21 (2010), pp. 1602-1611.

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